三模态报告基因的构建、体外标记和骨髓间充质干细胞显像的可行性
삼모태보고기인적구건、체외표기화골수간충질간세포현상적가행성
Novel triple fusion reporter gene for labeling and tracking human mesenchymal stem cells in vitro
  • 万方数据
    中华放射学杂志 중화방사학잡지
    Chinese Journal of Radiology
    2014年 9期 767-771 ,共5页

    秦潇潇%胡晓俊%李征然%陈俊伟%吴春%赖丽莎%张丽娜%谢佩怡%孟晓春

    진소소%호효준%리정연%진준위%오춘%뢰려사%장려나%사패이%맹효춘

    骨髓间充质干细胞%基因显像%对比研究 骨髓間充質榦細胞%基因顯像%對比研究
    골수간충질간세포%기인현상%대비연구
    Human bone marrow mesenchymal stem cells%Gene imaging%Comparative study
    目的 探讨三模态报告基因的构建、体外标记和人骨髓间充质干细胞(hMSC)显像的可行性.方法 通过gateway技术构建同时含有增强型绿色荧光蛋白(EGFP)、荧光素酶(luciferase2)和储铁蛋白(ferritin)为报告基因的慢病毒载体pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP.基因测序检测基因序列的正确性、慢病毒包装、感染hMSC,建立稳定的hMSC转染细胞系.以未转染hMSC为未转染组,转染hMSC为转染组.2组细胞分别应用荧光显微镜检测细胞中EGFP表达,光学成像系统计数细胞中加入催化底物D-luciferin后Is内发光光子数,普鲁士蓝染色检测铁染色率,利用MR的T2-mapping序列检测细胞的T2值.采用Pearson法分析转染组细胞个数与1s内发光光子数的相关性;采用t检验比较转染组和未转染组之间细胞的铁染色率、MR T2-mapping序列T2值,及转染加铁组与未转染加铁组之间T2值的差异.结果 基因测序显示pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP基因序列正确.慢病毒成功制备并转染hMSC,建立EGFP+-hMSC稳定细胞系.荧光显微镜可检测到单个转染组细胞EGFP表达,未转染组细胞中未检测到EGFP的表达.1ml不同浓度2.00×106、1.00× 106、5.00× 105、2.50× 105、1.25×105、6.25×104个/ml转染组细胞悬液产生的光子数分别为7.972× 107、3.061×1 07、1.547×107、7.805×106、4.256× 106、1.411×106,二者浓度呈正相关(r=0.993,P＜0.01).未转染组细胞中未检测到光学信号.普鲁士蓝染色可见转染组和未转染组中hMSC的铁染色率分别(81.6±5.1)％和(21.6±3.8)％,差异有统计学意义(t=20.97,P＜0.01).标记细胞MR成像,转染组细胞T2值为(628.0±23.1)ms,未转染组T2值为(646.5±17.2)ms,差异无统计学意义(t=1.286,P=0.246).转染加铁组T2值为(488.3±10.7) ms,明显低于未转染加铁组T2值[(520.8±21.0) ms],差异有统计学意义(t=2.76,P=0.033).结论 构建同时含有EGFP、luciferase2和ferritin基因的三模态基因显像方法可行,并可成功导入hMSC基因组中,在子代hMSC中共同高效表达.
    목적 탐토삼모태보고기인적구건、체외표기화인골수간충질간세포(hMSC)현상적가행성.방법 통과gateway기술구건동시함유증강형록색형광단백(EGFP)、형광소매(luciferase2)화저철단백(ferritin)위보고기인적만병독재체pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP.기인측서검측기인서렬적정학성、만병독포장、감염hMSC,건립은정적hMSC전염세포계.이미전염hMSC위미전염조,전염hMSC위전염조.2조세포분별응용형광현미경검측세포중EGFP표체,광학성상계통계수세포중가입최화저물D-luciferin후Is내발광광자수,보로사람염색검측철염색솔,이용MR적T2-mapping서렬검측세포적T2치.채용Pearson법분석전염조세포개수여1s내발광광자수적상관성;채용t검험비교전염조화미전염조지간세포적철염색솔、MR T2-mapping서렬T2치,급전염가철조여미전염가철조지간T2치적차이.결과 기인측서현시pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP기인서렬정학.만병독성공제비병전염hMSC,건립EGFP+-hMSC은정세포계.형광현미경가검측도단개전염조세포EGFP표체,미전염조세포중미검측도EGFP적표체.1ml불동농도2.00×106、1.00× 106、5.00× 105、2.50× 105、1.25×105、6.25×104개/ml전염조세포현액산생적광자수분별위7.972× 107、3.061×1 07、1.547×107、7.805×106、4.256× 106、1.411×106,이자농도정정상관(r=0.993,P＜0.01).미전염조세포중미검측도광학신호.보로사람염색가견전염조화미전염조중hMSC적철염색솔분별(81.6±5.1)％화(21.6±3.8)％,차이유통계학의의(t=20.97,P＜0.01).표기세포MR성상,전염조세포T2치위(628.0±23.1)ms,미전염조T2치위(646.5±17.2)ms,차이무통계학의의(t=1.286,P=0.246).전염가철조T2치위(488.3±10.7) ms,명현저우미전염가철조T2치[(520.8±21.0) ms],차이유통계학의의(t=2.76,P=0.033).결론 구건동시함유EGFP、luciferase2화ferritin기인적삼모태기인현상방법가행,병가성공도입hMSC기인조중,재자대hMSC중공동고효표체.
    Objective To evaluate the feasibility of labeling and tracing human bone marrow mesenchymal stem cells (hMSC) by a triple fusion reporter gene imaging.Methods A lentivirus vector,the pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP,which contained optical reporter gene-enhanced green fluorescent protein (EGFP),bioluminescent reporter gene-luciferase2 and MR reporter gene-ferritin,was constructed by gateway technology.After the accuracy of gene sequence was validated,the lentivirus was packaged and infected hMSC,and a stable transformed hMSC cell line was established by fluorescence-activated cell sorter.The expression of EGFP was inspected with fluoresence microscope; the bioluminescence photon number was counted by optical imaging system in 1 s after D-luciferin was given; the iron content of the cells was detected by prussian blue iron stain; T2 values of the T2-mapping of MRI were measured.After an extended series of 3 to 5 times repeated experiment,results were compared between transfected hMSC and un-transfected hMSC to explore the efficiency of the labeling and tracking hMSC of the reporter genes.The positive rate of expression of the EGFP,the difference in iron stain and MR signal (T2) between the two groups was measured by t test.The bioluminescence photon detected in two gradient groups was count to set a Pearson correlation analysis.Results The gene sequence of pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP was confirmed by DNA sequencing analysis.The lentivirus vector was successfully constructed and infected hMSC.A stable transformed hMSC cell line,with triple fusion reporter genes,was successfully established for the follow-up test.Single hMSC expressed EGFP could be detected by fluorescence microscope.The optical signal of 2.00× 106,1.00× 106,5.00× 105,2.50× 105,1.25 × 102,6.25 × 104/ml cells could be detected as 7.972× 107,3.061 × 107,1.547 × 107,7.805 × 106,4.256 × 106,1.411 × 106 bioluminescence photon after D-luciferin was given,and there was linear positive correlation between the intensity of optical signal and the number of cells (r=0.993,P＜0.01),while no optical signal was captured under the same experimental condition.The iron stain rate of transfection cells was (81.6± 5.1)％,which was significantly higher than that of un-transfection cells (21.6±3.6)％ (t=20.97,P＜0.01).MR showed the difference of T2 value of 2.5× 106 cells per milliliter between the transfection (628.0±23.1)ms andun-transfection cells (646.5±17.2)ms,but the difference was not significant (t=1.286,P=0.246).However,the difference was obvious when ammonium ferric citrate was given,(488.3± 10.7)ms in the transfeetion cells and (520.8 ± 21.0)ms in the un-transfected cells (t=2.76,P=0.033).Conclusion A triple fusion reporter gene imaging,including EGFP,luciferase2 and ferritin,is feasible,which can successfully be transfected into hMSC and effectively expressed in the daughter cells.
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